Activation of the innate immune response by bacteria is mediated by a group of molecules that have collectively been termed modulins. These surface-associated molecules are produced by a broad array of microbial Monocytes, macrophages, neutrophils and epithelial cells respond to modulins by producing proinflammatory cytokines. The production of cytokines is essential for protective innate and adaptive immune responses. However, excessive production of cytokines has also been implicated in the pathologic events associated with the septic response in patients with severe bacterial infections. We and others have demonstrated that flagella from a number of gram-negative bacterial species induce proinflammatory cytokine production by monocytes and intestinal epithelial cells. We demonstrated that the major structural protein of the flagella, flagellin, is the component required for the induction of cytokine synthesis. Flagellin signaling is mediated by a toll-like receptor (TLR) protein, TLR5, but can be modulated by another TLR, TLR4. In view of the extraordinary potency of flagellin as an activator of cells that are involved in host defense, we propose the following specific aims: 1. To determine the components and pathways that are required for TLR5-dependent flagellin signal transduction. GG2EE is a macrophage cell line derived from the LPS-hyporesponsive C3H/HeJ mouse. Like the cells from this mouse strain, GG2EE cells express a defective form of TLR4 and thus respond poorly to LPS. In contrast, GG2EE cells express a wild-type form of TLR5 and thus produce TNFa in response to flagellin. However, these cells do not produce nitric oxide in response to flagellin. We will determine the TLR requirements and signaling components for flagellin signaling leading to cytokine and nitric oxide synthesis. 2. To determine the mechanism by which flagellin induces a subsequent state of flagellin tolerance. Responsiveness to flagellin can be influenced by several factors including flagellin itself. Cells that are exposed to flagellin become insensitive to secondary stimulation by flagellin. This lack of responsiveness is associated with a dramatic decrease in IRAK kinase activity. This decrease in IRAK activity is not due to a decrease in the level of the IRAK protein. Other investigators have established that exposure of monocytes to LPS also results in a state of LPS non-responsiveness. LPS tolerant cells also exhibit a very low level of flagellin responsiveness. However, flagellin tolerant cells are fully responsive to LPS. We will determine the mechanism for this effect of flagellin since it has important implications with regard to the host response to flagellin. Furthermore, the proposed studies should also provide insights into the factors that regulate TLR signaling and allow for complex patterns of host cell response.